Reemergence of pathogenic, autoantibody-producing B cell clones in myasthenia gravis following B cell depletion therapy

Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. A small subset of patients (<10%) with MG, have autoantibodies targeting muscle-specific tyrosine kinase (MuSK). MuSK MG patients respond well to CD20-mediated B cell depletion therapy (BCDT); most achieve complete stable remission. However, relapse often occurs. To further understand the immunomechanisms underlying relapse, we studied autoantibody-producing B cells over the course of BCDT. We developed a fluorescently labeled antigen to enrich for MuSK-specific B cells, which was validated with a novel Nalm6 cell line engineered to express a human MuSK-specific B cell receptor. B cells (≅ 2.6 million) from 12 different samples collected from nine MuSK MG patients were screened for MuSK specificity. We successfully isolated two MuSK-specific IgG4 subclass-expressing plasmablasts from two of these patients, who were experiencing a relapse after a BCDT-induced remission. Human recombinant MuSK mAbs were then generated to validate binding specificity and characterize their molecular properties. Both mAbs were strong MuSK binders, they recognized the Ig1-like domain of MuSK, and showed pathogenic capacity when tested in an acetylcholine receptor (AChR) clustering assay. The presence of persistent clonal relatives of these MuSK-specific B cell clones was investigated through B cell receptor repertoire tracing of 63,977 unique clones derived from longitudinal samples collected from these two patients. Clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-mediated remission, predating disease relapse by several months. These findings demonstrate that a reservoir of rare pathogenic MuSK autoantibody-expressing B cell clones survive BCDT and reemerge into circulation prior to manifestation of clinical relapse. Overall, this study provides both a mechanistic understanding of MuSK MG relapse and a valuable candidate biomarker for relapse prediction. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-022-01454-0.


Reemergence of pathogenic, autoantibody-producing B cell clones in myasthenia gravis
Supplement Figure 9 Distance-to-nearest plots used to identify the threshold required for assigning clonal members in the BCR sequencing data. Table 1 Study subject clinical, laboratory, and demographic data.

Supplement Table 2
Radioimmunoassay-based testing of the 2E6 and 6C6 mAbs.

Supplement Table 3
Characteristics and analysis status of serial samples from patients MuSK MG-1 and MuSK MG-4.

Supplement Table 4
Counts of reconstructed V(D)J sequences by isotype and clones from sequencing of bulk BCR repertoires and 10x.

Supplement Figure 2
Supplement Figure 2. Flow cytometry gating strategy for isolation of MuSK-specific B cells. A representative example of the gating strategy featuring the fluorescently labeled MuSK ectodomain reagent is shown. After B cell enrichment using negative selection beads, single cells were gated using the forward (FSC) and side (SSC) scatter. Dead cells were excluded, then CD3 neg CD14 neg CD19 + IgD neg CD27 + IgM neg MuSK-reagent + cells were single cell sorted for subsequent B cell culture and expansion. Figure 3. Cell-based assay contour plots showing dilution series of mAbs 2E6 and 6C6. Binding to MuSK was tested over a series of ten two-fold dilutions of each mAb ranging from 10-0.02 µg/ml. The x-axis represents GFP fluorescence intensity and, consequently, the fraction of HEK cells transfected with MuSK. The y-axis represents Alexa Fluor 647 fluorescence intensity, which corresponds to the secondary anti-human IgG Fc antibody binding and, consequently, primary antibody binding to MuSK. Hence, transfected cells are located in the right quadrants and cells with MuSK antibody binding in the upper quadrants. The MuSK-specific human mAb MuSK1A was used as a positive control and the AChR-specific human mAb 637 used as a negative control.

Supplement Figure 4. Staining murine neuromuscular junctions with MuSK mAbs 2E6 and 6C6.
Immunofluorescent staining of mouse neuromuscular junctions (NMJ). Tibialis anterior muscles were cut longitudinally in cryosections and fixed with PFA. AChRs were stained with Alexa Fluor 648 αbungarotoxin (shown in red). The mAb 637 was used a positive control, to identify the location of the AChR. Binding of mAbs was detected with goat anti-human IgG Alexa Fluor 488 (IgG, shown in green).

Supplement Figure 5. Binding properties of unmutated common ancestors from MuSK mAbs 2E6
and 6C6. Representative cell-based assay (CBA) contour plots are shown (left) for the unmutated common ancestors (UCA) of 2E6 and 6C6. The x-axis represents GFP fluorescence intensity and, consequently, the fraction of HEK cells transfected with MuSK. The y-axis represents Alexa Fluor 647 fluorescence intensity, which corresponds to secondary anti-human IgG Fc antibody binding and, consequently, primary antibody binding to MuSK. Hence, transfected cells are located in the right quadrants and cells with MuSK antibody binding in the upper quadrants. The plots show testing with a mAb concentrations of 10 and 1.25 µg/ml. Binding to MuSK was tested over a series of ten two-fold dilutions of each mAb ranging from 10-0.02 µg/ml (right). The MuSK1A mAb was used as the positive control and AChR-specific mAb 637 as the negative control. The ∆MFI was calculated by subtracting the signal acquired by testing non-transfected cells from the signal acquired by testing transfected cells. Each data point represents the mean value from three independent experiments. Symbols represent means and error bars SDs. Values greater than the mean + 4SD of the negative control mAb at 1.25 µg/ml (indicated by the horizontal dotted line) were considered positive. Supplement Figure 9. Distance-to-nearest plots used to identify the threshold required for assigning clonal members in the BCR sequencing data. Distance-to-nearest plots used to identify a common threshold to use for hierarchical clustering-based grouping of V(D)J sequences from high throughout sequencing of BCR repertoires. Red dashed lines correspond to the threshold used for assigning clonal clusters. Grey bars represent the distribution of intra-subject distance-to-nearest distances.  Table 3. Characteristics and analysis status of serial samples from patient MuSK MG-1 and MuSK MG-4. These longitudinally collected samples were used for investigating whether clones or clonal variants of mAbs 6C6 and 2E6 were present. Antibody titer was either measured by CBA in our laboratory or at the Mayo Clinic Laboratory. Samples of MuSK MG-4 measured at Mayo Clinic Laboratory are indicated by an (*); the unit is nmol/L. The cut off for negativity for samples measured at Mayo Clinic Laboratory is ≤ 0.02 nmol/L. The autoantibody titers of MuSK MG-1 measured by our CBA was performed using 10 two-fold dilutions ranging from 1:20 to 1:10240. TOC = time of collection; Pred = Prednisone